Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 101 bp
Wild Type = 118 bp
>chr4:150149636+150149753 118bp GCTTCCACTTTCTTCCAGGAG TCCAGGTTCCCCTACACTGA
Sequence (deleted region in lower case)
ACCAGATACAACCTCTTCACAACCCTACCTGTCCCTACACCTTCAAGGTCAAAAGCAGGAAAAATGTGTCCCTAGAAACAGGATAACACCTGCTTGGGTCACCTTCCCAGACAGCCCCCTCCTCCTCCTTCCAGGATTAGAGCCTGCTCCCAGCCAGACCTCCCTTTCCACctaaagaggaagtggaggcctggccaccctcaccccaccccaatcctagaagtgacaggagccaaccctacagagtctctgacctcgaaactctctggttcagcgtctgcagcccacactagtcctgactaccctaagtgctgcctttggctcagtcttccagtatggctacaacatcgccgtgatcaacacacctcacaaggtgggcacaaggtgggtctcagcgggtagaagtcagctgctgtctggcttctggaagcttccactttcttccaggagtcccctagatacctacccctaaggactaggggcatctagagccatTTTATCTAGCAATGTCATCTTTTCTAGGGGTATCAGTGTAGGGGAACCTGGATTCTCTGCCTGGGCACCCTGCCTTGCTCACTTGGGAAGGCCATTTGTGGGTGACAGACACATCAAGAATGAACTGTTAACTTTTAGAGATTGTGTTTGGTCAAAGAGAAGCAACAGAGATAAAGGCTGACTGGAGCAAGTTCA
324 bp deletion beginning at Chromosome 4 positive strand position 150,149,378 bp and ending after 150,149,701 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37747 | TCC TCC TTC CAG GAT TAG AGC | Mutant Forward | A | |||
37748 | TCC AGG TTC CCC TAC ACT GA | Common | A | |||
37749 | GCT TCC ACT TTC TTC CAG GAG | Wild type Forward | A | |||
37750 | Fluorophore-1 | CCA GCC AGA CCT CCC TTT | Quencher-1 | MUT Probe | ||
37751 | Fluorophore-2 | CTA CCC CTA AGG ACT AGG GGC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37747 | 0.40 uM |
37748 | 0.40 uM |
37749 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |